Expression of DNA sequences encoding a thermally stable cytosine deaminase from saccharomyces

Abstract

Thermally stable cytosine deaminase (CDase), and the gene coding therefor, is disclosed as well as methods of isolating, purifying, and recombinantly producing the same. The thermally stable CDase can be isolated from Saccharomyces cerevisiae. The yeast isolated enzyme has a molecular weight of approximately 32 kDa, as determined by gel filtration chromatography, and is composed of two subunits, each with a molecular weight of about 17 kDa. Thermally stable yeast CDase so purified shows no significant sequence homology with other known sequenced proteins.

We claim: 
     
       1. An isolated nucleotide sequence encoding a thermally stable Saccharomyces cytosine deaminase having the protein sequence depicted in FIG. 10. 
     
     
       2. A recombinant expression vector comprising and effective in expressing the nucleotide sequence of claim 1. 
     
     
       3. The recombinant expression vector of claim 2 wherein the nucleotide sequence is operably linked to control sequences compatible with a procaryotic host. 
     
     
       4. The recombinant expression vector of claim 2 wherein the nucleotide sequence is operably linked to control sequences compatible with a eucaryotic host. 
     
     
       5. The recombinant expression vector of claim 4 wherein the nucleotide sequence is operably linked to control sequences compatible with a mammalian host. 
     
     
       6. Bacterial cells transformed with the recombinant expression vector of claim 3. 
     
     
       7. Mammalian cells transformed with the recombinant expression vector of claim 5. 
     
     
       8. A method of producing recombinant, thermally stable cytosine deaminase, or a protein functionally equivalent thereto, comprising growing the bacterial cells of claim 6. 
     
     
       9. A method of producing recombinant, thermally stable cytosine deaminase, or a protein functionally equivalent thereto, comprising growing the mammalian cells of claim 7.